This application relates to a method for detection and identification of microorganisms, including in particular pathogenic microorganisms, and to compositions and kits useful in practicing the method. The invention can be applied to detection of viruses, including HIV and hepatitis, bacteria, including Chlamydia, fungi, including Cryptococcus neoformans and protozoa, including Trypanosoma cruzi. This application also relates to DNA sequencing reactions, and in particular to improved bi-directional sequencing reaction protocols making use of thermally stable polymerase enzymes.
Detection of the presence of pathogenic microorganisms through DNA-based technology is emerging as an important tool in the diagnosis of many diseases. For example, diagnosis of Chlamydia trachomatis infections, the most common bacterial sexually transmitted disease in North America, is shifting from traditional methods such as culture, enzyme immunoassay (EIA) and direct fluorescent antibodies (DFA) to DNA-hybridization diagnostics. Roche Diagnostic Systems, Inc. (Nutley, N.J.) manufactures Amplicor.TM., a test which detects C. trachomatis and Neisseria gonorrohoeae by the hybridization of a pathogen specific probe to PCR amplified products, detectable by a color change/optical density technique. Abbott Laboratories (Abbott Park, Ill.) makes UriProbe, also a test for C. trachomatis and N. gonorrohoeae, which relies on the ligase chain reaction (LCR). The LCR method, described in Patent Applications WO 9320227, WO 9300447, WO 9408047, WO 9403636, EP 477 972 uses thermostable ligase enzyme to ligate two DNA probes which hybridize in ligatable juxtaposition on a template DNA strand, thus generating a detectable ligated DNA fragment only if the template DNA is present. A multiplex PCR assay for C. trachomatis has also been described in Mahony et al., J. Clin. Microbiol. 33: 3049-3053 (1995).
The ideal DNA sequencing procedure for use in a diagnostic environment would have the following characteristics: (1) it would be able to utilize a DNA-containing sample which had been subjected to only minimal pretreatment to make the DNA accessible for sequencing; (2) it would require combining this sample with only a single reaction mixture, thus reducing risk of error and contamination, and increasing the ease with which the procedure can be automated; and (3) it would require a short amount of time to perform the sequence determination, thus decreasing the marginal costs in terms of equipment and labor for performing the test.
A wide variety if infectious pathogens that can be detected by DNA-based methods are listed in Diagnostic Molecular Microbiology, Persing et al., eds. American Society for Microbiology, Washington D.C. (1993). This text details diagnostic tests for bacteria, virus, fungi, and protozoa. Diagnostic tests are also proposed for identifying the presence of drug resistance genes or toxin genes.
Although these tests are generally effective for identifying an infectious disease-causing organism if present, they do not routinely provide information concerning the specific serotype, variant or form of the infecting organism. Depending on the organism in question, this information can be significant in determining the likely course of the infection, for determining the most appropriate therapeutic approach and for epidemiological purposes. Furthermore, the previously known assays involve several steps and are therefore more susceptible to systematic error than would be a test with fewer steps. Thus, there remains a need for a simple test format which is generally applicable to the detection of microorganisms, including infectious disease-causing microorganisms, and particularly for a simple test which provides an indication of the specific nature, e.g., the serotype, of the organism. It is an object of the present invention to provide such a test.
It is an object of the present invention to provide reagent combinations useful in performing tests for infectious disease-causing microorganisms, including Chlamydia human papilloma virus(HPV) and HIV.
It is a further object of the present invention to provide kits useful in performing tests for infectious disease-causing microorganisms, including Chlamydia, HPV and HIV.
It is an additional object of the present invention to provide an improved method for bi-directional sequencing of DNA samples which is well-suited for use in the diagnostic environment and for automation.
It is an additional object of the invention to provide a method for bi-directional sequencing of DNA which utilizes a DNA-containing sample which has been subjected to only minimal pretreatment to make the DNA accessible for sequencing.
It is still a further object of the invention to provide a method for bi-directional sequencing of DNA which requires combining a complex DNA-containing sample with only a single reaction mixture, thus reducing risk of error and contamination, and increasing the ease with which the procedure can be automated.